Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. Conclusion: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.

Addition of dextran sulfate to blood cardioplegia attenuates reperfusion injury in a porcine model of cardiopulmonary bypass

OBJECTIVE: Contact of blood with artificial surfaces and air as well as ischemia/reperfusion injury to the heart and lungs mediate systemic and local inflammation during cardiopulmonary bypass (CPB). Activation of complement and coagulation cascades leads to and accompanies endothelial cell damage. Therefore, endothelial-targeted cytoprotection with the complement inhibitor and endothelial protectant dextran sulfate (DXS, MW 5000) may attenuate CBP-associated myocardial and pulmonary injury. METHODS: Eighteen pigs (DXS, n=10; phosphate buffered saline [PBS], n=8) underwent standard cardiopulmonary bypass. After aortic cross-clamping, cardiac arrest was initiated with modified Buckberg blood cardioplegia (BCP), repeated after 30 and 60 min with BCP containing either DXS (300 mg/10 ml, equivalent to 5mg/kg) or 10 ml of PBS. Following 30 min reperfusion, pigs were weaned from CPB. During 2h of observation, cardiac function was monitored by echocardiography and invasive pressure measurements. Inflammatory and coagulation markers were assessed regularly. Animals were then sacrificed and heart and lungs analyzed. RESULTS: DXS significantly reduced CK-MB levels (43.4+/-14.8 ng/ml PBS, 35.9+/-11.1 ng/ml DXS, p=0.042) and significantly diminished cytokine release: TNFalpha (1507.6+/-269.2 pg/ml PBS, 222.1+/-125.6 pg/ml DXS, p=0.0071), IL1beta (1081.8+/-203.0 pg/ml PBS, 110.7+/-79.4 pg/ml DXS, p=0.0071), IL-6 (173.0+/-91.5 pg/ml PBS, 40.8+/-19.4 pg/ml DXS, p=0.002) and IL-8 (304.6+/-81.3 pg/ml PBS, 25.4+/-14.2 pg/ml DXS, p=0.0071). Tissue endothelin-1 levels were significantly reduced (6.29+/-1.90 pg/100mg PBS, 3.55+/-1.15 pg/100mg DXS p=0.030) as well as thrombin-anti-thrombin formation (20.7+/-1.0 microg/ml PBS, 12.8+/-4.1 microg/ml DXS, p=0.043). Also DXS reduced cardiac and pulmonary complement deposition, neutrophil infiltration, hemorrhage and pulmonary edema (measured as lung water content, 81+/-3% vs 78+/-3%, p=0.047), indicative of attenuated myocardial and pulmonary CPB-injury. Diastolic left ventricular function (measured as dp/dt(min)), pulmonary artery pressure (21+/-3 mmHg PBS, 19+/-3 mmHg DXS, p=0.002) and right ventricular pressure (21+/-1 mmHg PBS, 19+/-3 mmHg DXS p=0.021) were significantly improved with the use of DXS. CONCLUSIONS: Addition of DXS to the BCP solution ameliorates post-CPB injury and to a certain extent improves cardiopulmonary function. Endothelial protection in addition to myocyte protection may improve post-CPB outcome and recovery.

Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Macrophage-independent T cell infiltration to the site of injury-induced brain inflammation

We have addressed the role of macrophages in glial response and T cell entry to the CNS after axonal injury, by using intravenous injection of clodronate-loaded mannosylated liposomes, in C57BL6 mice. As expected, clodronate-liposome treatment resulted in depletion of peripheral macrophages which was confirmed by F4/80- and MOMA-1(-) stainings in spleen. Sequential clodronate-liposome treatment 4, 2 and 0 days before axotomy resulted in significant reduction of infiltrating CD45(high) CD11b+ macrophages in the hippocampus at 1, 2 and 3 days post-lesion, measured by flow cytometry. There was a slight delay in the expansion of CD45(dim) CD11+ microglia in clodronate-liposome treated mice, but macrophage depletion had no effect on the percentage of infiltrating T cells in the lesion-reactive hippocampus. Lesion-induced TNFalpha mRNA expression was not affected by macrophage depletion, suggesting that activated glial cells are the primary source of this cytokine in the axonal injury-reactive brain. This identifies a potentially important distinction from inflammatory autoimmune infiltration in EAE, where macrophages are a prominent source of TNFalpha and their depletion prevents parenchymal T cell infiltration and disease.

1.59 Reduced inflammatory potential of circulating gammadelta T cells in pregnancy induced improvement of Rheumatoid Arthritis

BACKGROUND AND OBJECTIVES During pregnancy, gammadelta T cells expand at the fetomaternal interface where they induce a tolerogenic milieu. Patients with rheumatoid arthritis (RA) experience a spontaneous improvement of their disease during pregnancy and a postpartum aggravation. By contrast, pregnant patients with ankylosing spondylitis (AS) often experience persistent active disease. We hypothesised that the pregnancy related modulation of disease activity in RA patients versus AS patients is associated with numerical and functional changes of circulating gammadelta T cells. MATERIAL AND METHODS The frequency of surface markers and the intracellular cytokine profile of freshly isolated gammadelta T cells from RA (n = 54) and AS (n = 26) patients and healthy controls (n = 40) were analysed at each trimester during pregnancy and 6-8 weeks postpartum by flow cytometry. RESULTS Very discrete changes of Vdelta1 or Vdelta2 frequency were seen during pregnancy and postpartum in healthy controls and AS patients. In RA, however, the frequency of Vdelta2 cells decreased in the third trimester when disease activity was low. Low percentages of Vdelta 2 cells were also found in non-pregnant RA patients with active arthritis, yet only pregnant RA patients showed reduced percentages of Vdelta2 cells positive for the activation marker CD69 and the intracellular cytokine TNFalpha. Similarly, Vdelta1 + TNFalpha + cells were lower in pregnant RA patients compared to non-pregnant RA patients. The percentage of Vdelta2 + TNFalpha + cells, Vdelta2+ CD69+ and Vdelta1+ CD69+ cells correlated with disease activity in RA. As for the receptors which modulate cytotoxicity, RA patients showed a rise of the anti-cytotoxic receptor NKG2A on Vdelta1 cells in the 2(nd) trimester and a decrease postpartum. Since the pro-cytotoxic receptor NKG2D remained unchanged, the NKG2D/NKG2A ratio on Vdelta1 cells was reduced in RA patients during pregnancy. In AS patients, persistent disease activity during pregnancy was reflected by an increased frequency of Vdelta2+ CD69+ cells and an unchanged frequency of Vdelta2+ TNFalpha+ cells. In addition, pregnant AS patients showed an increased frequency of Vdelta1+CD161+ cells. CONCLUSIONS Disease amelioration of RA during pregnancy correlates with changes of cell activation, pro-inflammatory cytokines and anti-cytotoxic receptors of gammadelta T cells. By contrast, active disease during pregnancy as found in AS is associated with unchanged inflammatory responses of gammadelta T cells. Since gammadelta T cells remain unchanged in healthy pregnant controls, the modulation of gammadelta T cells in RA rather seems to be an effect of improved disease than of pregnancy itself.

Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

IKKalpha mediated NOTCH1 Activation Promotes Tumorigenesis via FOXA2 Suppression

Proinflammatory cytokine TNFa plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFa-mediated tumor development remains unclear. Here, we show that IKKa, an important downstream kinase of TNFa, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKa, pFOXA2 (S107/ 111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKa and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFa/IKKa-associated FOXA2 inhibition likely contributes to inflammationmediated cancer pathogenesis. Here, we report a TNFa/IKKa/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.

IRS-1 regulation in health and disease

The global incidence of diabetes is increasing at epidemic rates. Estimates suggest there are currently 150 million people with diabetes and this number is expected to double in the next 20 years. Type 2 diabetes accounts for 95% of all cases and is characterized in part by impaired sensitivity to insulin or 'insulin resistance'. Defects in the insulin signalling pathways underpin this resistance. In the current article we discuss the regulation of Insulin Receptor Substrate-1 (IRS-1), a protein that plays a pivotal role in insulin signalling and whose function is impaired in subjects with insulin resistance. Coordination of IRS-1 function is multi-faceted, involving phosphorylation of IRS-1 at multiple serine/threonine residues. This controls many aspects of IRS-1, including its interaction with the insulin receptor and subsequent tyrosine phosphorylation, as well as its subcellular distribution and targeting for degradation by the proteasome. Such tight control ensures appropriate transduction and attenuation of the insulin signal, thereby regulating insulin action in healthy individuals. Emerging evidence indicates that `diabetogenic factors' associated with insulin resistance, such as TNFalpha and elevated circulating fatty acids, impact on insulin signalling at the level of IRS-1 serine/threonine phosphorylation. The expression and/or activity of several kinases, such as IkappaB kinase beta (IKKbeta) and salt-induced kinase 2 (SIK2), and the phosphorylation of IRS-1 at key sites, such as Ser307 and Ser789, are increased in states of insulin resistance. Identifying the pathways by which such factors activate these and other kinases, and de. ning the precise roles of specific serine/threonine phosphorylation events in IRS-1 regulation, represent important goals which may eventually provide a rationale for therapeutic intervention.

Physical activity, exercise, and inflammatory markers in older adults: Findings from the health, aging and body composition study

OBJECTIVES: To examine the association between physical activity and inflammatory markers, with consideration for body fatness and antioxidant use. DESIGN: Cross-sectional study, using baseline data from the Health, Aging and Body Composition Study. SETTING: Metropolitan areas surrounding Pittsburgh, Pennsylvania, and Memphis, Tennessee. PARTICIPANTS: Black and white, well-functioning men and women (N=3,075), aged 70 to 79. MEASUREMENTS: Interviewer-administered questionnaires of previous-week household, walking, exercise, and occupational/volunteer physical activities. Analysis of covariance was used to examine the association between activity level and serum C-reactive protein (CRP), interleukin-6 (IL-6), and plasma tumor necrosis factor alpha (TNFalpha) with covariate adjustment. Antioxidant supplement use (multivitamin, vitamins E or C, beta carotene) was evaluated as an effect modifier of the association. RESULTS: Higher levels of exercise were associated with lower levels of CRP (P

S100A8 chemotactic protein is abundantly increased, but only a minor contributor to LPS-induced, steroid resistant neutrophilic lung inflammation in vivo

Neutrophilic lung inflammation is an essential component of host defense against diverse eukaryotic and prokaryotic pathogens, but in chronic inflammatory lung diseases, such as chronic obstructive lung disease (COPD), severe asthma, cystic fibrosis, and bronchiolitis, it may damage the host. Glucocorticosteroids are widely used in these conditions and in their infectious exacerbations; however, the clinical efficacy of steroids is disputed. In this study, we used a proteomic approach to identify molecules contributing to neutrophilic inflammation induced by transnasal administration of lipopolysaccharide (LPS) that were also resistant to the potent glucocorticosteroid dexamethasone (Dex). We confirmed that Dex was biologically active at both the transcript (suppression of GM-CSF and TNFalpha transcripts) and protein levels (induction of lipocortin) and used 2D-PAGE/MALDI-TOF to generate global expression profiles, identifying six LPS-induced proteins that were Dex resistant. Of these, S100A8, a candidate neutrophil chemotactic factor, was profiled in detail. Steroid refractory S100A8 expression was highly abundant, transcriptionally regulated, secreted into lung lavage fluid and immunohistochemically localized to tissue infiltrating neutrophils. However, in marked contrast to other vascular beds, neutralizing antibodies to S100A8 had only a weak anti-neutrophil recruitment effect and antibodies against the related S100A9 were ineffective. These data highlight the need for extensive in vivo profiling of proteomically identified candidate molecules and demonstrates that S100A8, despite its abundance, resistance to steroids and known chemotactic activity, is unlikely to be an important determinant of LPS-induced neutrophilic lung inflammation in vivo.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.